With the ever increasing need to acquire more biological analytes from cells, biological protocols are becoming increasingly more complex. For instance, to capture both RNA and DNA from a sample including a single population of cells, a process that is desired but difficult with existing technology, a first buffer is necessary to lyse the cell membrane to expose the RNA. Thereafter, a second buffer must be combined with the first buffer to lyse the nuclear membrane, exposing the DNA. Additionally, different buffer conditions (e.g., salt concentrations) are necessary to promote the capture of RNA and DNA to magnetic beads. Hence, in order to sequentially extract RNA and DNA from a single sample, additional buffer must be added following the extraction of RNA to facilitate sequential DNA capture. Thus, strategies are needed to enable this “buffer addition” that are compatible with existing extraction techniques.
With the advent of Exclusion-based Sample Preparation (ESP), a simplified sample preparation process, droplets with convex menisci have proven advantageous. In ESP, the volumes of droplets of the samples/reagents are locked into specific narrow ranges. Hence, to accommodate variable sample/reagent volumes, multiple wells are needed. Each well has its own narrow volume range, thereby enabling users to utilize only the wells appropriate for each application. It can be appreciated that in order to do sequential capture of RNA and DNA, the droplets must be diluted. However, it is not possible to dilute a droplet of sufficient volume in a well that is already filled to a convex shape.
Currently, there are two options for buffer addition. First, following RNA removal, the remaining sample volume can be transferred to a new well, where an additional buffer is added. Unfortunately, this option requires sample transfer, which can lead to analyte loss, particularly for rare analytes. Second, the second buffer may be added directly to the initial sample well following RNA extraction. However, because the meniscus is already convex, filling the well further with the second buffer will likely cause the well to overflow. As a result, the sample may be lost.
Therefore, it is a primary object and feature of the present invention to provide a method and a device for containing expanding droplets at predetermined locations along a surface of a microfluidic device.
It is a further object and feature of the present invention to provide a method and a device for containing expanding droplets at predetermined locations along a surface along a microfluidic device that enables the droplets to be continuously enlarged, while maintaining a prescribed perimeter and a convex meniscus.
It is a further object and feature of the present invention to provide a method and a device for containing expanding droplets at predetermined locations along a surface of a microfluidic device that is simple to utilize and inexpensive to manufacture.
It is a still further object and feature of the present invention to provide a method and a device for containing expanding droplets at predetermined locations along a surface of a microfluidic device that is compatible with current sample preparation processes.
In accordance with the present invention, a microfluidic device is provided. The microfluidic device includes a plate having an upper surface and a central region communicating with the upper surface. The central region is adapted for receiving a droplet of fluid thereon. The central region includes an outer periphery that defines a first fluid constraint configured for discouraging fluid on the central region from flowing therepast. A second fluid constraint extends about the first fluid constraint. The second fluid constraint is configured for discouraging fluid flowing therepast. A third fluid constraint extends about the second fluid constraint. The third fluid constraint is configured for discouraging fluid flowing therepast.
The central region may include a recess formed in the upper surface of the plate. The recess is defined by a closed bottom spaced from the upper surface by a first sidewall. The first sidewall intersects the upper surface at a first edge. The first edge defines the first fluid constraint. The plate may also include a first channel in the upper surface. The first channel extends about the first sidewall and is defined by a first recessed surface spaced from the upper surface by a second sidewall. The second sidewall intersects the upper surface at a second edge. The second edge defines the second fluid constraint. The plate may also include a second channel in the upper surface. The second channel extends about the second sidewall and is defined by a second recessed surface spaced from the upper surface by a third sidewall. The third sidewall intersects the upper surface at a third edge. The third edge defines the third fluid constraint. The first channel has a volume. The volume of the first channel is greater than a volume of the second channel.
Alternatively, the first fluid constraint may include a first hydrophobic ring extending along the outer periphery of the central region and the second fluid constraint may include a second hydrophobic ring extending about the first fluid constraint. The second hydrophobic ring is radially spaced from the first hydrophobic ring. The third fluid constraint includes a third hydrophobic ring extending about the second fluid constraint. The third hydrophobic ring is radially spaced from the second hydrophobic ring.
In the alternative, a first sidewall may have a first end intersecting the outer periphery of the central region at a first edge and a second end. The first edge defines the first fluid constraint. A first ledge extends radially from the second end of the first sidewall and terminates at a terminal first edge. The terminal first edge defines the second fluid constraint. A second sidewall depends from the terminal first edge and terminates at a lower end. A second ledge extends radially from the lower end of the second sidewall and terminates at a terminal second edge. The terminal second edge defines the third fluid constraint.
In accordance with a further aspect of the present invention, a device is provided for containing a droplet having an outer surface at a predetermined location. The droplet has an internal pressure. The device includes a microfluidic device having a surface and a first fluid constraint extending about a first droplet area for receiving the droplet therein. The first fluid constraint is configured for maintaining the droplet within the first droplet area in response to the internal pressure of the droplet failing to exceed a first threshold. A second fluid constraint extends about and is spaced from the first fluid constraint by a second droplet area for receiving the droplet thereon. The second fluid restraint is configured for maintaining at least a portion of the droplet within the second droplet area in response to the internal pressure of the droplet failing to exceed a second threshold. A third fluid constraint extends about and is spaced from the second fluid constraint by a third droplet area for receiving the droplet thereon. The third fluid restraint is configured for maintaining at least a portion of the droplet within the third droplet area in response to the internal pressure of the droplet failing to exceed a third threshold.
The first droplet area may include a recess formed in the surface of the microfluidic device. The recess is defined by a closed bottom spaced from the surface by a first sidewall. The first sidewall intersects the surface at a first edge. The first edge defines the first fluid constraint. A first channel may be formed in the upper surface. The first channel extends about the first sidewall and is defined by a first recessed surface spaced from the upper surface by a second sidewall. The second sidewall intersects the surface at a second edge. The second edge defines the second fluid constraint. A second channel may be formed in the surface. The second channel extends about the second sidewall and is defined by a second recessed surface spaced from the surface by a third sidewall. The third sidewall intersects the surface at a third edge. The third edge defines the third fluid constraint. The first channel has a volume. The volume of the first channel may be generally equal to a volume of the second channel.
Alternatively, the first, second and third fluid constraints may be defined by corresponding concentric hydrophobic bands radially spaced from each other along the surface of the microfluidic device. In a further alternative, a fluid retainer extends from the surface of the microfluidic device and is defined by a plurality of steps such that the fluid retainer has a stepped pyramid configuration. Each step includes a rise generally perpendicular to the surface and a landing generally parallel to the surface wherein the intersection of each rise and landing combination defines a corresponding one of the first, second and third fluid constraints.
In accordance with a still further aspect of the present invention, a method is provided for containing an expandable droplet at a predetermined location along a surface of a microfluidic device. The method includes the steps of providing a plurality of radially spaced fluid constraints about a droplet deposit region of the surface of the microfluidic device and depositing a droplet within on the droplet deposit region of the surface of the microfluidic device. The droplet is retained within a first fluid constraint of the plurality of radially spaced fluid constraints if an internal pressure of the droplet is less than a first threshold. The droplet is retained within a second fluid constraint of the plurality of radially spaced fluid constraints if the internal pressure of the droplet is less than a second threshold. The droplet is retained within a third fluid constraint of the plurality of radially spaced fluid constraints if the internal pressure of the droplet is less than a third threshold.
The droplet deposit region may include a recess formed in the surface of the microfluidic device. The recess may include by a closed bottom spaced from the surface by a first sidewall. The first sidewall intersects the surface at a first edge. The first edge defines the first fluid constraint. A first channel may be provided in the upper surface. The first channel extends about and is radially spaced from the first sidewall and is defined by a first recessed surface spaced from the surface of the microfluidic device by a second sidewall. The second sidewall intersects the surface at a second edge. The second edge defines the second fluid constraint. A second channel may be provided in the surface. The second channel extends about and is radially spaced from the second sidewall and is defined by a second recessed surface spaced from the surface by a third sidewall. The third sidewall intersects the surface at a third edge. The third edge defines the third fluid constraint. The first channel has a volume. The volume of the first channel may be generally equal to a volume of the second channel.
Alternatively, the first, second and third fluid constraints may be defined by corresponding concentric hydrophobic bands radially spaced from each other along the surface of the microfluidic device. In a further alternative, a fluid retainer extends from the surface of the microfluidic device and is defined by a plurality of steps such that the fluid retainer has a stepped pyramid configuration. Each step includes a rise generally perpendicular to the surface and a landing generally parallel to the surface wherein the intersection of each rise and landing defines a corresponding one of the first, second and third fluid constraints.